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K L Yielding

Publications and source records attributed to K L Yielding.

At least 37 records · Page 2Linked to original sources
PubMed1984

Ethidium binding sites on plasmid DNA determined by photoaffinity labeling.

Photoaffinity labeling of pBR322 with ethidium monoazide (8-azido-3-amino-5-ethyl-6-phenylphenanthridinium chloride) was used to provide evidence for the sequence specificity of ethidium binding to native DNA. DNA-drug interactions were examined at concentrations of eight covalently bound ethidium drugs per molecule of pBR322 (4363 base pairs). Restriction enzyme cutting was blocked by the covalent binding of a drug molecule at (or near) the enzyme recognition sequence. This phenomenon was observed with all restriction enzymes tested and was not limited to specific regions of the pBR322 molecule. Double-digestion experiments indicated that a drug molecule may bind 2 to 3 base pairs outside the recognition sequence and still block restriction enzyme digestion. Intact plasmid was treated with [3H]ethidium monoazide and digested with restriction enzymes. The amount of covalently-linked ethidium analog was quantitated for different restriction fragments and the G-C content of each fragment was determined from the DNA sequence. In approximately half of the fragments the drug appeared to preferentially bind at a G-C base pair. However, no preference for specific sequences such as 5'-C-G-3' was detected, as had been suggested by previous modeling studies with ethidium bromide. The other fragments were located in specific map regions of the plasmid and did not bind drug with a strict dependence on GC content suggesting that binding specificity may depend on more than one structural feature of the DNA.

Affinity Labels↗
PubMed1984

DNA damage and repair in mouse embryos following treatment transplacentally with methylnitrosourea and methylmethanesulfonate.

Mouse embryos were labeled in vivo at 10 1/2-12 1/2 days of gestation with [3H]-thymidine and subjected to DNA damage using x-ray, methylmethanesulfonate, or methylnitrosourea. DNA damage and its repair were assessed in specific cell preparations from embryos isolated at intervals thereafter using the highly sensitive method of nucleoid sedimentation, which evaluates the supercoiled state of the DNA. Repair of x-ray damage was demonstrated using trypsin-dispersed cells from whole embryos and from homogenized embryonic liver to show the validity of the analytical approach. The effects of the highly teratogenic methylnitrosourea and the much less teratogenic methylmethanesulfonate were compared in the targeted limb buds using equitoxic doses of the two alkylating agents. DNA supercoiling was fully restored after 24 hr in limb bud cells damaged with methylmethanesulfonate, while as much as 48 hr were required for full repair of methylnitrosourea damage. These results demonstrated the feasibility of studying DNA repair in embryonic tissues after damage in vivo and suggest that the potency of methylnitrosourea as a teratogen may be correlated with a prolonged period required for complete repair of DNA.

Animals↗
PubMed1984

Petite and sectored induction in Saccharomyces cerevisiae by propidium iodide: synergistic effect of sodium dodecyl sulfate.

Sodium dodecyl sulfate (SDS) was examined for its effect on petite and sectored colony induction in Saccharomyces cerevisiae by propidium iodide (PI) and ethidium bromide (EB). 4-h cultivation with 100 microM PI and 100 micrograms/ml SDS resulted in virtually all plated cells growing as sectored colonies with no decrease in viability. Sectored colonies are mixed colonies comprised of respiratory deficient and competent cells believed to be derived from an unstable respiratory deficient cell. Further cultivation with PI and SDS prior to plating led to induction of complete petite colonies with a rapid decrease in viable cells. PI alone at this concentration exhibited weak induction of sectored colonies (maximum 12.3% at 8 h) and petite colonies (maximum 10.8% at 12 h), but SDS alone caused induction of neither. 50 microM PI had almost the same activity as 100 microM except for a delay in the induction of sectored colonies in the initial stage, and a decreased rate of petite colony induction. The effects of 20 microM PI and SDS were much lower than that by 50 microM and no inhibition of growth was observed. 10 microM PI was quite inactive even in the presence of SDS. Under resting conditions, 10 approximately 100 microM PI and 100 micrograms/ml SDS induced about 60% sectored colonies at 12 h incubation and more than 60% petite colonies at 24 h. After 6 h incubation, decrease in survival was also observed.

Antimycin A↗
PubMed1984

Induction of cytoplasmically inherited respiration-deficient ('petite') mutants by photodynamic action of acridine compounds.

All acridines used (acriflavine, proflavine, acridine orange and 3-azido-10-methylacridinium chloride) produced killing in yeast cells when activated with visible light. Acriflavine, proflavine and 3-azido-10-methylacridinium chloride, but not acridine orange, produced petite and sectored colonies. Both cell killing and petite induction by light activation of acriflavine resulted apparently from photodynamic action mediated by singlet oxygen (1O2) since the effect were prevented by either sodium azide or anaerobiosis. The biological effects of 3-azido-10-methylacridinium chloride, which was developed as a potential photoaffinity probe for studying the binding and biological effects of acridines, appeared to be due to a photodynamic action analogous to that of acriflavine. Sodium azide or anaerobiosis prevented the light-activated effects of 3-azido-10-methylacridinium chloride despite the fact that the initial chemical breakdown of the azido derivative induced by light was not affected. Cells suspended in D2O demonstrated an enhanced response to 3-azido-10-methylacridinium chloride with irradiation. These results indicate that singlet oxygen mediates the light-activated biological effects of both acriflavine and 3-azido-10-methylacridinium chloride.

Acridines↗
PubMed1984

Petite induction in Saccharomyces cerevisiae by ethidium analogs. Action on mitochondrial genome.

Petite induction of ethidium analogs was examined in both resting and growing yeast cells. All of the analogs used in these experiments were active in dividing cells of Saccharomyces cerevisiae; only the parent ethidium bromide was mutagenic under resting conditions. Incorporation of adenine into mitochondrial DNA appeared to be prevented completely by ethidium and partially inhibited by other analogs. Treatment of growing cells with analogs affected fragmentation of pre-existing DNA as seen by the loss of a mitochondrial antibiotic resistance marker. The rates of elimination of the marker were different; ethidium generated greater loss than the monoamino analogs (3-amino and 8-amino-); and the deaminated analog was least effective. However, in resting yeast the marker was partially eliminated only with treatment of the parent ethidium. The degradation of the mitochondrial DNA by exposure to ethidium compounds was confirmed by agarose gel electrophoresis. Electrophoretic patterns of the mitochondrial DNA treated with each of the analogs under growing conditions and only with ethidium under resting conditions showed degradation of the mitochondrial DNA.

Cell Cycle↗
PubMed1984

Observations on epidermal exsorption in mice following injections of procion dyes and ethidium bromide and topically applied dimethyl sulfoxide.

Back skin of hairless mice and external ears of CD-1 white mice were used to study the details of epidermal exsorption . Ethidium bromide, a DNA ligand, and two dichlorotriazinyl (procion) dyes were injected, i.v. or i.p. Migration patterns from the skin vasculature into the epidermis were observed by fluorescence microscopy and microspectrofluorometry . Topically applied DMSO greatly enhanced the exsorption process and produced intensely labeled epidermis. Ethidium bromide reacted primarily with nuclear DNA of living cells while the procion dyes tended to migrate intercellularly to label the stratum corneum. External ears of white mice treated topically with DMSO showed a pattern of labeling which included intense fluorescence of the cartilage and perichondrium as well as the ear epidermis.

Administration, Topical↗
PubMed1984

Ultrastructural studies of barrier restoration in epidermis of hairless mice following dimethyl sulfoxide application.

Sixteen hairless mice were studied by transmission electron microscopy to determine the ultrastructural changes in epidermal barrier restoration following topical applications of dimethyl sulfoxide (DMSO) to back skin. Samples of experimental skin were examined at 30 min, 1 h, 2 h and 3 h after the initial DMSO treatment and compared with that of control animals. At earlier time periods (30 min-1 h) the DMSO treated epidermis showed greatly expanded intercellular spaces, disrupted desmosomal attachments, and desmosomal remnants lying free within the intercellular space. Mitochondria contained droplets of lipoid material which reached maximum size in the spinous and granular layers. Cytoplasmic vacuoles were particularly prominent in the 30-min samples. Large numbers of membrane coating granules (MCG) emerged in the 1 h specimens. The MCG were laden with numerous electron-dense inner membranes which crisscrossed at various angles. Many of the MCG were observed in the process of fusion with plasma membranes of granular cells adjacent to the stratum corneum. At 2 h a process of recovery from the effects of DMSO was evident and at 3 h there was little or no difference between experimental and control epidermis.

Animals↗
PubMed1984

Subcellular localization of photoreactive ethidium analogs in Trypanosoma brucei by fluorescence microscopy.

To identify the in vivo targets of the trypanocide, ethidium bromide, the fluorescent staining of T. brucei was examined for a series of ethidium analogs using fluorescence microscopy. Determination of the biological targets for most drugs is limited by the reversible nature of their interactions. To overcome this limitation, photoaffinity (azido) analogs of ethidium, which are capable of covalent attachment with photoactivation, were used to identify the ethidium binding sites within the parasites. Two of these compounds, when covalently attached, demonstrated an enhancement of fluorescent staining and were selective for the kinetoplast at low drug concentrations. These compounds were also those found previously to have the highest trypanocidal activity. Propidium, a phenanthridinium analog identical to ethidium except for a larger, more ionic substitution at R5, showed more nonspecific binding as determined by its general staining of the cytoplasm.

Affinity Labels↗
PubMed1982

Demonstration of specific high affinity binding sites in plasmid DNA by photoaffinity labeling with an ethidium analog.

We have used photoaffinity labeling of pBR322 DNA with 8-azido-3-amino-5-ethyl-6-phenylphenanthridinium chloride to demonstrate high affinity ethidium-binding sites. Plasmid equilibrated with as little as 1 drug/DNA molecule was photoactivated, freed of uncomplexed drug by ethanol precipitation, and subjected to restriction analysis. There was highly specific, rather than random, blockage of HhaI sites (d(GCGC)) at low drug concentrations. Furthermore, the same 7 new digestion fragments were generated at drug to nucleotide ratios ranging from 1:100 to 1:8000. All the new DNA fragments had chain lengths greater than the largest HhaI fragment (393 base pairs). At higher ligand concentrations closely approximating those needed for equilibrium binding studies, detection of the high affinity sites was greatly masked. Drug binding to HhaI restriction fragments which had been prepared prior to the action of drug did not induce new bands. Furthermore, the larger DNA fragments from drug-labeled plasmid were resistant to HhaI digestion over a wide range of enzyme concentrations. These findings suggest that ligand binding can be highly selective even between sites which have the same tetranucleotide sequence. Therefore, selective drug binding must be dictated not only by local base sequence preference, but also by other long range parameters.

Affinity Labels↗
PubMed1982

Focal cerebellar dystrophy caused by transplacental administration of methylnitrosourea.

Methylnitrosourea (MNU), 20 mg/kg, was given IP to CD-1 mic on day 16 of pregnancy and the offspring examined at 3 and 5 weeks of age. In addition to a general reduction in brain size in all specimens, there were focal alterations in cerebellar architecture. Specifically, the granule cells of the anterior lobe and vermis were reduced in number and ectopic in localization. There were concomitant changes in the localization of the Purkinje cells suggesting changes in migration influences. These experiments used a short-lived (15 minute), direct-acting DNA alkylating agent to produce focal cerebellar damage. MNU therefore, appears to be a promising tool for examining regional developmental abnormalities in the central nervous system.

Aging↗
PubMed1981

Comparison of petite induction in yeast by acridines, ethidium and their photoaffinity probes.

The production of petite mutations by different acridine analogs was studied in Saccharomyces cerevisiae. Compounds with amino substituents at the 2 and 3 positions of the acridine nucleus and methylation at position 10 were effective for petite induction in growing cells but not in resting cells, while those with chloro, nitro and methoxy substituents were not effective in either resting or growing cells. Photosensitive azido derivatives of the acridines were tested to evaluate the role of covalent drug attachment for mutagenesis in resting cells. Photolysis of resting cells with 9-axido, 3-azido-6-amino-, 9-azido-10-methyl-, or 3-azido-6-amino-10-methyl-acridine was highly toxic. 3-Azido-6-amino-acridine, and especially 3-azido-10-methyl-, and 3-azido-6-amino-10-methyl-acridine, were effective petite inducers in resting cells. Thus, the photosensitive (azido) group at position 9 produced only cell killing while the azido group at position 3 and/or 6 led to effective petite induction in resting cells. In contrast, petite induction was observed only for growing cells, for dark control experiments with these compounds or with the monoazide precursor compounds.

Acridines↗
PubMed1981

Induction of petite "mutants" in an ethidium-resistant strain of Saccharomyces cerevisiae by photoaffinity labeling. Distinction between early and late steps.

A strain of Saccharomyces cerevisiae (MH41-7B/011) was resistant to petite induction by ethidium bromide at 30 degrees, but was sensitive to induction by photolabeling with ethidium monoazide. These results suggested a defect in the mutant in metabolic activation of ethidium to account for its resistance. Synchronized cultures of both the mutant and the normal parent strains showed a substantial reduction in petite response to photolabeling in stationary phase cells which could not be accounted for by changes in cell penetration of the drug. The use of photolabeling with normal and mutant cells suggested that petite induction can be divided into early and late steps.

Drug Resistance, Microbial↗
PubMed1981

Antitrypanosomal action enhanced by photoaffinity labeling with ethidium analogs.

The trypanocidal activity of photoreactive azido analogs of ethidium was tested to determine the suitability of using such compounds as in vivo probes to study the mechanism of the antitrypanosomal activity of ethidium. Eight ethidium analogs, including three nonphotoreactive compounds, were tested for their ability to kill T. brucei both with and without photolytic activation. Two analogs tested, the monoamino-monoazido isomers, showed greater that 100-fold enhancement of trypanocidal activity following photolytic activation in situ. Without photolytic activation, only the nonphotoreactive monoamino precursor analogs showed activity greater than the parent ethidium compound. The availability of suitable ethidium analogs which can be covalently attached by in situ photoactivation provides a new approach for studying the mechanism by which ethidium exerts its trypanocidal activity.

Animals↗
PubMed1980

Binding of ethidium monoazide to the chromatin in human lymphocytes.

The azide analog of [14C]ethidium bromide was mixed with lymphocytes and photolyzed with visible light. The distribution of azide in the chromatin fraction was found to be 55% in DNA, 28% in protein and 16% in RNA. Label in the DNA portion was found to be almost exclusively in the region digestible with micrococcal nuclease. The parent compound, ethidium bromide, competed with azide for binding sites, illustrating that the azide analog mimics the action of ethidium bromide.

Affinity Labels↗
PubMed1980

Co-mutagenic effects of propidium on petite induction by ethidium in Saccharomyces cerevisiae.

Propidium, whose structure is closely related to ethidium bromide, induced a low level of petites in yeast, but only at high concentrations with long incubation time, and only in growth medium. When added to growing cells, propidium also caused a large increase in petite induction by ethidium even at submutagenic concentrations of ethidium. Incorporation of adenine into DNA was inhibited by propidium in mitochondria but not in nuclei. Propidium by itself had no effects on fragmentation of pre-existing DNA, but enhanced mitochondrial DNA degradation provoked by ethidium. The proportion of suppressive clones occurring among the petites from ethidium treatment was reduced by the presence of propidium. All of these results indicated that propidium treatment led to degradation of the mitochondrial DNA in petites induced by ethidium but not in native (intact) mitochondrial DNA, nor in spontaneous petite colonies. The results are discussed in terms of possible mechanisms of modulation of petite induction.

Adenine↗
PubMed1980

Petite induction in Saccharomyces cerevisiae by ethidium analogs: distinction between resting and growing cells.

The importance of specific substituents, especially amino azide groups, for ethidium induction of petites was evaluated in resting and dividing cells of Saccharomyces cerevisiae through the study of a series of ethidium analogs. The structural requirements in resting and growing cells were found to be different, suggesting that at least two mechanisms are responsible for induction. The significance of particular substituents in the induction processes were recognized by: (1) a dependence upon the ethyl substituent at the ring nitrogen in both actively growing and in resting cells; and (2) the implication that amino substituents are important for the effect in dividing cells and especially in resting cells. Photolytic enhancement of petite induction (via a nitrene which forms a covalent linkage to a biological site) was observed for 3 of the azide analogs, which emphasizes the likelihood that metabolic activation of ethidium to a covalent complex is responsible for its effectiveness. Furthermore, these studies indicate that these monoazide analogs should be ideal probes for examining the mitochondrial mutagenic processes.

Ethidium↗